pGEX-2t purification problem - (Sep/01/2013 )
I have my gene cloned into pGEX-2t , I changed everything temp, concentration IPTG, Combatant cells but protein still insoluble.
I do not know if using Urea can destroy my protein or not cause I will do methyltransferase assy in this protein. Is using L- Arginine with the medium that will help solubleization?. I am wondring if anyone used this before .
Many thanks for you help
Which lysis buffers have you tried? Detergents? Salt concentrations? denaturing agents? glycerol?
I have used PBS and sonication then I add triton 1%
Try a range of salt concentrations from 500 mM to 100 mM, addition of 2-ME and glycerol can help sometimes, as can trying different detergents.